As part of the UIUC Neurometabolomics and Neuroproteomics Center on Cell-Cell Signaling, the Sampling and Separation Core develops and applies methods to enhance all aspects of sample preparation and fractionation for the comprehensive chemical analysis of brain neurochemistry at the tissue, cell, and subcellular levels. Precise, accurate, and careful sampling is crucial in successful proteome, peptidome, and metabolome studies. In contrast to DNA/RNA-based molecular techniques, direct measurement of proteins and metabolites in volume- and mass-limited samples lacks a significant signal amplification step. This Core offers unmatched capabilities in the sampling of tissues, cells, and organelles, enabling the chemical characterization of various classes of molecules from an ever-increasing range of samples and animal models. Infused throughout this Core is the philosophy that directed sampling is fundamental to reducing the intrinsic chemical and morphological complexity of heterogeneous neuronal systems to achieve effective measurement of dynamic brain systems. The methodological advances we have developed already provide better results for experiments that, in some cases, would have been considered impossible to perform without these approaches. A unifying strategy for studies employing separation methods is that they seek to fractionate complex tissue samples into components that make them more accessible for in-depth chemical characterization via mass spectrometry analysis in the Molecular Profiling and Characterization Core. Since our inception, we have been expanding our analytical toolset to include a number of both well-established and innovative approaches for analyte sampling and separation, thus providing robust processing of nervous system samples for metabolite, peptide, and protein characterization. Obviously, this Core is closely linked to the needs of our users, and exists within the framework of the entire Center. Only when integrated with the Pilot Research Projects Core, the Molecular Profiling and Characterization Core, and the Bioinformatics, Data Analytics, and Predictive Modeling Core, are we able to generate the biological information required by our users. Simply stated, the Sampling and Separation Core links the biological user base to the rest of the Center and lays the foundation for successful Center to user project outcomes. The close interactions between the neuroscience users and the Sampling Core chemists and biologists enables a robust synergy. The neuroscience users have become increasingly aware of the advantages provided by the analytical tools and expertise available to them through our Core. So too have the facility chemists gained greater familiarity with the diversity of sample types, from a honey bee brain to a rat brain, with sample sizes ranging from whole brain extracts to single cells, to subcellular compartments. We tightly couple the methodologies to the neuroscience to ensure that we will continue to advance analytical methods of significant value to the broader NIDA research community.